Rash Laboratory

Fig. 1 Confocal images of Mauthner cells labeled for Cx35 and glutamate receptor NR1, with companion FRIL immunogold labeling of gap junctions in goldfish Mauthner cells.

(A) Confocal immunofluorescence image (three Z sections of 2 µm) showing Cx35 (MAB 3045) at a Club Ending on distal portion of a Mauthner Cell lateral dendrite. These terminals, identified by their large size, exhibit multiple sites of punctate labeling for Cx35. (B and C) Cx35 (Ab298) labeling of conventional “plaque” gap junctions (red overlay) in MC/CE synapses. Both images are from the same goldfish MC/CE synapse, showing primarily postsynaptic P-face (B) and presynaptic E-face (C). In both images, all labeling is on postsynaptic connexins. (D) Laser scanning immunofluore-scence image (three Z sections of 2 µm) of a Club Ending on a Mauthner-cell lateral dendrite after labeling with anti-NR1 antibody. (E–H) Simultaneous co-localization of Cx35 at gap junctions and NR1 glutamate receptors in E-face particles arrays (yellow overlay) in goldfish Mauthner cell. (E) E-face view of the postsynaptic membrane of goldfish Mauthner cell. E-face image of gap junction in postsynaptic membrane, but with unfractured and unreplicated presynaptic plasma membrane beneath. It is these unreplicated presynaptic connexins that are labeled (18 nm gold). In contrast, the two arrays of E-face IMPs represent glutamate receptor clusters, which are immunogold labeled for NR1 (12 nm gold beads in the extracellular space; see Fig. 2B–E for explanatory diagram). Inscribed areas are shown at higher magnification (F–H). (F) Stereoscopic (left pair) and reverse stereoscopic images (right pair) of a neuronal gap junction labeled for Cx35. (G and H) Portions of two PSDs after immunogold labeling for glutamate receptor NR1 by two gold beads (G) and one gold bead (H). The gap junction and the PSD on the right are separated by~50 nm, or less than the limit of resolution of light microscopy. For confocal images A and D, calibration bars = 5 µm. In all FRIL replicas, calibration bars = 0.1 µm, unless otherwise designated.

Fig. 2 Immunogold labeling of glutamate receptor PSDs and nearby gap junctions.

(A) Portions of three Cx35-immunogoldlabeled gap junctions (red overlays) surrounding a P-face imprint of a PSD (yellow overlay), corresponding to the arrays of IMPs on E-faces (see Fig. 1G and H). The PSD P-face pits are devoid of NR-1 labeling. (B–E) Drawing of fracture plane through two Club Ending synapses on a Mauthner cell. (B and C) From left to right, the fracture plane (blue line) sequentially fractures within the postsynaptic plasma membrane, through a glutamate receptor PSD (lavender) and through a postsynaptic
gap junction (left side of image), across the Mauthner cell cytoplasm (center of image), then to within the Mauthner cell plasma membrane (right side), exposing the postsynaptic membrane E-face particles corresponding to glutamate receptors, then the gap junction E-face pits (postsynaptic membrane) and, finally, gap junction P-face particles (presynaptic membrane of lower club ending). (B and C) PSD P-face pits are not labeled because no glutamate receptor proteins remain with the P-face pits (left side). However, P-face connexon IMPs (orange connexons, left side) in the adjacent gap junction are labeled. Glutamate receptor E-face particles are labeled on their extracellular determinants (right side), whereas connexons of presynaptic gap
junctions are labeled on their cytoplasmic epitopes (D and E). In the gap junction on the right (2C, box) and in closer view (D), postsynaptic E-face pits and presynaptic P-face particles are replicated, but in both cases, it is the presynaptic connexons (yellow connexons) of the underlying Club Ending that are labeled. (E) Diagram showing immunogold labeling of epitope in
the cytoplasmic carboxy terminus of a single connexin molecule by Ab298 (Pereda et al., 2003). (F) Stereoscopic and reverse stereoscopic image of portion of mixed synapse in adult rat inferior olive labeled for Cx36 (20 nm gold; red overlay) and NR1 glutamate receptors (10 nm gold, arrow; yellow overlay). Reverse stereoscopy (right pair) is helpful for discerning the 10 nm gold bead superimposed on the equally electron-dense platinum replica, as well as for discriminating between the two layers of replica that arose when a portion of replica was displaced (darker area at top).

Fig. 3  Stereoscopic images of gap junctions in oligodendrocyte somatic plasma membranes after double-labeling for Cx47 and Cx32.

(A) One large gap junction (three arrowheads delineating its edge) exhibits robust labeling for Cx47 (136 12nm gold) but little or no labeling for Cx32 (no 6nm and only one 18nm gold bead). N, nucleus; *, cytoplasm. The lower left quadrant of the image is not shadowed with platinum because of blockade by a large tissue fragment, but instead is replicated by carbon only. (B) Higher magnification stereoscopic image of the same gap junction. Carbon-replicated connexons are faintly delineated, whereas platinum-shadowed connexons (upper right) are clearly delineated. White arrowhead, 18nm gold bead for Cx32. Unlabeled scale bars=0.1 μm.

Fig. 4  Gap junctions on oligodendritic processes in sample of rat spinal cord after single-labeling for Cx47 (12nm gold beads).

(A) Cytoplasmic process (“oligodendrite”) linking an oligodendrocyte soma (not shown) to the outermost layer of myelin (M) of two different cross-fractured axons (Ax). Arrow indicates area of transition from dendritic process to flattened cytoplasmic myelin. Of seven gap junctions on this dendritic process (arrowheads), all were heavily labeled for Cx47. (B) Stereoscopic image of two closely-adjacent gap junctions (or one irregular gap junction), from the inscribed area in 4A; labeled by 20 12nm gold beads. (C) One or two small gap junctions on the same dendrite; labeled for Cx47 by eight 12nm gold beads. (D) Large (>1500 connexons), medium and small gap junctions on oligodendrite; labeled for Cx47. Two central “reciprocal patches,” which are composed of mixed IMPs and pits, are not labeled. Unlabeled scale bars=0.1 μm.

Fig. 5  Gap junctions on the outer surface of myelin that had been double-labeled for Cx47+Cx32.

(A–F) Myelin is characterized by broad expanses of particle free membranes. Gap junctions are distinguished from “reciprocal patches” (RP in A) by having 100% of IMPs on P-faces and 100% of pits on E-faces. Plasma membranes of astrocytes were identified by the presence of E-face images of AQP4 square arrays (C; white arrow). (C) Enlarged image of B. (B) M, cross-fractured myelin. (A–C) Most gap junctions (66%) were immunogold labeled for Cx47 (12nm gold) but did not label for Cx32. A few (21%), particularly the smallest gap junctions (D), were double-labeled for Cx47 (12nm gold beads) plus Cx32 (white arrowhead, 6nm gold bead; black arrowhead, 18nm gold bead), and fewer still (13%) on outer myelin (E, F) were labeled for Cx32, only. Scale bars = 0.1 μm.

Fig. 6  Laser scanning confocal double immunofluorescence images of caspr and Cx32 labeling in white matter of adult rat spinal cord.

(A) Low magnification images of dorsolateral white matter showing intermittent labeling for caspr along myelinated fibers (A1, arrowheads), intermittent (A2, arrowheads) as well as continuous (A2, arrows) labeling for Cx32 along these fibers, and occasional regions of overlap (A3, arrowheads). (B) Higher magnification images from regions similar to those in (A) showing granular appearance of labeling for Cx32 along some fibers (B2, arrows), and intense immunofluorescence for caspr (B1, arrowheads) and Cx32 (B2, arrowheads) on each side of a node of Ranvier. Overlay shows overlap of labeling (B3, arrowheads). (C–E) Double immunofluorescence labeling showing co-localization of caspr and Cx32 (arrowheads) in other white matter regions of spinal cord, including ventrolateral (C), ventral (D), and dorsal columns (E). Scale bars = 25 μm A; B–E, 5 μm.

Fig. 7  FRIL images of gap junctions at Schmidt-Lanterman incisure; between the innermost tongue of cytoplasmic myelin and the second innermost layer of partially-compacted myelin; immunogold labeled for Cx32 (6nm and 18nm gold) and Cx47 (12nm gold, none present).

(A) M, myelin; E, E-face; P, P-face; white arrow, astrocyte process containing bundle of GFAP filaments; black arrow, cytoplasm of innermost tongue of myelin. Inscribed areas shown at higher magnification, below. (B) *, cytoplasm of 2nd and 3rd innermost layers of myelin. (C–E) High magnification stereoscopic images of three gap junctions labeled for Cx32 (from small inscribed boxes in A). (C′–E′) Reverse stereoscopic images of C–E allow recognition of 6nm gold beads against the equally electron-dense platinum replica. White arrowhead, 6nm gold beads for Cx32; black arrowhead, 18nm gold bead for Cx32. (C″–E″) Reversed contrast (i.e. black shadows) versions of C–E, which more clearly reveal the hexagonal arrays of E-face pits (black pits or holes representing where connexons had been removed; black shadows according to the original imaging convention of Steere 1957). Nearby E-face IMPs cast distinctive black shadows, as if the IMPs had been illuminated with white light. However, immunogold beads appear anomalously white. Unlabeled scale bars = 0.1 μm.

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